One of the major controversies of contemporary medicine is created by an increased consumption of nicotine and growing evidence of its connection to cancer, which urges elucidation of the molecular mechanisms of oncogenic effects of inhaled nicotine.
Chernyavsky et al BMC Cancer (2015) 15:152 DOI 10.1186/s12885-015-1158-4 RESEARCH ARTICLE Open Access Mechanisms of tumor-promoting activities of nicotine in lung cancer: synergistic effects of cell membrane and mitochondrial nicotinic acetylcholine receptors Alex I Chernyavsky1, Igor B Shchepotin2, Valentin Galitovkiy1 and Sergei A Grando1,3,4* Abstract Background: One of the major controversies of contemporary medicine is created by an increased consumption of nicotine and growing evidence of its connection to cancer, which urges elucidation of the molecular mechanisms of oncogenic effects of inhaled nicotine Current research indicates that nicotinergic regulation of cell survival and death is more complex than originally thought, because it involves signals emanating from both cell membrane (cm)- and mitochondrial (mt)-nicotinic acetylcholine receptors (nAChRs) In this study, we elaborated on the novel concept linking cm-nAChRs to growth promotion of lung cancer cells through cooperation with the growth factor signaling, and mt-nAChRs — to inhibition of intrinsic apoptosis through prevention of opening of mitochondrial permeability transition pore (mPTP) Methods: Experiments were performed with normal human lobar bronchial epithelial cells, the lung squamous cell carcinoma line SW900, and intact and NNK-transformed immortalized human bronchial cell line BEP2D Results: We demonstrated that the growth-promoting effect of nicotine mediated by activation of α7 cm-nAChR synergizes mainly with that of epidermal growth factor (EGF), α3 — vascular endothelial growth factor (VEGF), α4 — insulin-like growth factor I (IGF-I) and VEGF, whereas α9 with EGF, IGF-I and VEGF We also established the ligand-binding abilities of mt-nAChRs and demonstrated that quantity of the mt-nAChRs coupled to inhibition of mPTP opening increases upon malignant transformation Conclusions: These results indicated that the biological sum of simultaneous activation of cm- and mt-nAChRs produces a combination of growth-promoting and anti-apoptotic signals that implement the tumor-promoting action of nicotine on lung cells Therefore, nAChRs may be a promising molecular target to arrest lung cancer progression and re-open mitochondrial apoptotic pathways Keywords: Bronchial epithelial cells, Lung cancer cells, Nicotinic acetylcholine receptors, Proliferation, Growth factors, Intrinsic apoptosis, Mitochondria * Correspondence: sgrando@uci.edu Department of Dermatology, University of California, 134 Sprague Hall, Irvine, CA 92697, USA Department of Biological Chemistry, University of California, 134 Sprague Hall, Irvine, CA 92697, USA Full list of author information is available at the end of the article © 2015 Chernyavsky et al.; licensee BioMed Central This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Chernyavsky et al BMC Cancer (2015) 15:152 Background One of the major controversies of contemporary medicine is created by an increased consumption of nicotine and growing evidence of its connection to cancer (reviewed in [1]) Nicotine can contribute in a variety of ways to cancer survival, growth, metastasis, resistance to chemotherapy, and create a tumor-supporting microenvironment, thus implementing a "second hit" that aggravates aberrant signaling and elicits survival and expansion of cells with genomic damage [1] The list of cancers reportedly connected to nicotine is expanding, and presently includes small- and non-small cell lung carcinomas as well as head and neck, gastric, pancreatic, gallbladder, liver, colon, breast, cervical, urinary bladder and kidney cancers ([1] and references therein) Once limited to cigarettes, cigars, pipe tobacco and chewing or spit tobacco, nicotine-containing products today come in more flavors, forms, shapes and sizes, and with more unproven health claims Electronic cigarettes (eCigs) that aerosolize nicotine without generating toxic tobacco combustion products are rapidly gaining acceptance as an alternative to conventional cigarettes with little knowledge regarding their biomedical effects [2-4] eCig use, or vaping, allows to achieve systemic nicotine concentration similar to that produced from traditional cigarettes [5] Although eCigs are generally recognized as a safer alternative to combusted tobacco products, there are conflicting claims about the degree to which these products warrant concern for the health of the vapers [6,7], and there is a risk of a second- and thirdhand exposure to nicotine from eCigs [8] Thus, there is an urgent need for elucidation of the molecular mechanism of oncogenic effects of inhaled nicotine to facilitate development and evaluation of safety measures for eCigs Nicotine can displace the autocrine and paracrine hormone-like molecule acetylcholine (ACh) from the nicotinic class of ACh receptors (nAChRs) expressed in lung cells due to its higher receptor-binding affinity ACh is produced practically by all types of human cells, and is remarkably abundant in the lung epithelium [9,10] Increasingly, a wider role for ACh in cell biology is being recognized, including proliferation, differentiation, apoptosis, adhesion and motility (reviewed in [11,12]) The final cellular response to ACh is determined by the delicate balance between the growthpromoting and inhibiting signals The extracellular pool of ACh is replenished by vesicular ACh transporter secreting the ACh-containing vesicles, whereas the intracellular pool is represented mainly by free cytoplasmic ACh [13,14] In human bronchioalveolar carcinoma cells, nicotine upregulates choline acetyltransferase and vesicular ACh transporter, thus increasing production and secretion of ACh [15] Nicotine also can upregulate Page of 12 nAChR expression [16], thus shifting ACh signaling in lung cells toward the nicotinic vs muscarinic physiological signaling pathways The nAChRs are classic representatives of superfamily of the ligand-gated ion channel pentameric receptor proteins composed of ACh binding α subunits and "structural" subunits Lung cells can express the α1, α2, α3, α4, α5, α6, α7, α9, α10, β1, β3, β2, β4, γ, δ and ε nAChR subunits [17-22] The differences in subunit composition determine the functional and pharmacological characteristics of the receptor pentamers formed, so that the net biological effect produced by a nicotinic agonist depends on the subtype of nAChR binding this ligand with the highest affinity While direct involvement of α7 nAChR has been documented in the pathophysiology of lung cancer [23], α9 nAChR is known to play an important role in breast cancer [24-26] Silencing of the expression of nAChR subunits and treatment with nAChR antagonists produce anti-tumor effects both in vitro and in vivo [15,25,27-32] The nAChR subunit proteins can physically associate with both protein kinases and protein tyrosine phosphatases in large multimeric complexes [33] Even a shortterm exposure to nicotine activates mitogenic signaling pathways involving signaling kinases [34] The nAChRs mediate the nicotine-dependent upregulation of genes contributing to progression of lung cancer [35-38] Current research, however, indicates that nicotinergic regulation of cell survival and death is more complex than originally thought The emerging picture is that a diversity of molecular signaling circuitries regulating cancer cell growth signifies cross-talk interactions between cell membrane (cm-)nAChRs and growth factor (GF) receptors (GFRs), and receptors to various other autocrine and paracrine mediators [1] Additionally, modulation of functional electron transport in mitochondria has been recently found to play an important role in implementing the nicotine action interfering with chemotherapy-induced apoptosis [39] Nicotine can permeate lung cells and activate the mitochondrial (mt-)nAChR subtypes found on the mitochondrial outer membrane of lung cells [40] Activation of these receptors may inhibit opening of mPTP, which can block the initial step of intrinsic apoptosis [41-44] The mPTP is a multi-component protein aggregate comprised by structural elements of the inner as well as outer mitochondrial membrane that form a non-specific pore permeant to any molecule of